Fragile X syndrome (FXS) is caused by a repression of the FMR1 gene that codes the Fragile X mental retardation protein (FMRP), an RNA binding protein involved in processes that are crucial for proper brain development. To better understand the consequences of the absence of FMRP, we analyzed gene expression profiles and activities of cortical neural progenitor cells (NPCs) and neurons obtained from FXS patients’ induced pluripotent stem cells (IPSCs) and IPSC-derived cells from FMR1 knock-out engineered using CRISPR-CAS9 technology. Multielectrode array recordings revealed in FMR1 KO and FXS patient cells, decreased mean firing rates; activities blocked by tetrodotoxin application. Increased expression of presynaptic mRNA and transcription factors involved in the forebrain specification and decreased levels of mRNA coding AMPA and NMDA subunits were observed using RNA sequencing on FMR1 KO neurons and validated using quantitative PCR in both models. Intriguingly, 40% of the differentially expressed genes were commonly deregulated between NPCs and differentiating neurons with significant enrichments in FMRP targets and autism-related genes found amongst downregulated genes. Our findings suggest that the absence of FMRP affects transcriptional profiles since the NPC stage, and leads to impaired activity and neuronal differentiation over time, which illustrates the critical role of FMRP protein in neuronal development.

Primary cilia direct cellular signaling events during brain development and neuronal differentiation. The primary cilium is a dynamic organelle formed in a multistep process termed ciliogenesis that is tightly coordinated with the cell cycle. Genetic alterations, such as ciliary gene mutations, and epigenetic alterations, such as post-translational modifications and RNA processing of cilia related factors, give rise to human neuronal disorders and brain tumors such as glioblastoma and medulloblastoma. This review discusses the important role of genetics/epigenetics, as well as RNA processing and post-translational modifications in primary cilia function during brain development and cancer formation. We summarize mouse and human studies of ciliogenesis and primary cilia activity in the brain, and detail how cilia maintain neuronal progenitor populations and coordinate neuronal differentiation during development, as well as how cilia control different signaling pathways such as WNT, Sonic Hedgehog (SHH) and PDGF that are critical for neurogenesis. Moreover, we describe how post-translational modifications alter cilia formation and activity during development and carcinogenesis, and the impact of missplicing of ciliary genes leading to ciliopathies and cell cycle alterations. Finally, cilia genetic and epigenetic studies bring to light cellular and molecular mechanisms that underlie neurodevelopmental disorders and brain tumors.

Human midbrain organoids (hMOs) are a powerful stem cell-derived model for studying human neurodevelopment and neurological disease in vitro; however, their broad adoption is limited by long culture times, high cost, and inter-batch variability. Cryopreservation offers a strategy to address these limitations by enabling long-term storage and reducing experimental heterogeneity, but its effectiveness depends on robust cryoprotective strategies. Here, we evaluated hMO cryopreservation using a standard 10% dimethyl sulfoxide (DMSO) protocol, alone and in combination with a panel of five ice recrystallization inhibitors (IRIs), including betaine-derived ammonium salts and N-aryl-d-gluconamides. Across all conditions, hMOs were successfully recovered following freeze–thawing, with preservation of overall structural integrity, including maintained morphology and reduced cellular debris. Importantly, post-thaw viability and cell recovery in the 10% DMSO condition were comparable to those of fresh controls, demonstrating that conventional DMSO-based cryopreservation is sufficient to maintain hMO survival and gross structural preservation. While supplementation with select IRIs, particularly N-ethyl betaine ethyl ester iodide (NEBEE-I), yielded modest improvements in viability relative to DMSO alone and a non-significant trend toward enhanced electrophysiological activity in multielectrode array recordings, these effects were secondary to the robust performance of the baseline DMSO protocol. Collectively, these findings establish standard 10% DMSO cryopreservation as an effective and broadly applicable method for hMO preservation, while suggesting that small-molecule IRIs may provide incremental benefits in selected outcome measures.